ABSTRACT

Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11 is poorly neutralized by subtype-matched and subtype C sera, even compared to other tier 3 viruses, and is also recognized poorly by V3/glycan-targeting monoclonal antibodies (MAbs). We found that sequence polymorphisms in the V3 loop and N-linked glycosylation sites contribute only minimally to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane-proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus but is commonly recognized in the context of an HIV-2 chimera, suggesting steric or kinetic hindrance of binding to MPER in the native envelope (Env). Mutations in the 253-11 MPER, which were previously reported to increase the lifetime of the prefusion Env conformation, affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.

IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time that the virus spends in the open conformation following CD4 binding. Interestingly, we found that these mutations affect the 253-11 envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in toward the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a tier 3 envelope spike.

INTRODUCTION

The HIV-1 envelope (Env) is the sole target of HIV-1-specific neutralizing antibodies (NAbs) and therefore an attractive vaccine target (1, 2). It has been proposed that HIV-1 has progressively become more resistant to neutralizing antibodies over time during the HIV-1 pandemic (3–5). An understanding of these highly neutralization-resistant viruses may be needed to develop a global HIV-1 vaccine capable of protecting against most, if not all, HIV-1 strains.

Neutralization-resistant viruses have evolved numerous mechanisms to evade antibody responses. One of the ways in which viruses evade the immune response is through the formation of a “glycan shield,” protecting underlying epitopes (6–10). The ability of the virus to substantially mutate its sequence and remain functional is also a critical component of the viral antibody evasion strategy (8, 11). HIV-1 also evades neutralization through conformational masking (12, 13). The envelope (Env) in its prefusion state exists largely as closed trimer spikes with key targets of neutralizing antibodies at least partially occluded. As examples, the V1/V2 region at the apex of the envelope aids in the occlusion of the V3 loop, neighboring protomers in the trimer spike restrict the angle of approach to the CD4 binding site (CD4-bs), and the membrane-proximal external region (MPER) is often partially sunk into the membrane (14–18). In this closed state, narrowly neutralizing antibodies are generally unable to bind and neutralize the virus (14). Additionally, HIV-1 expresses nonfunctional gp120/gp41 monomers or gp41 stumps lacking gp120 (19) that may serve as decoys to divert the antibody response and contribute to yet another immune evasion mechanism.

HIV-1 Env is a class I membrane fusion glycoprotein that undergoes conformational changes associated with binding to its receptor, CD4, to enable coreceptor binding and subsequent viral membrane fusion with the host cell (20, 21). It is becoming increasingly clear that the HIV-1 Env spike is dynamic and shifts between closed and open conformations in the prefusion state (14, 22, 23). During the transition of the Env trimer to the open state, the trimer rearranges, facilitating a shift of the V1/V2 loops to the perimeter of the structure (15, 22, 24, 25). In the transient and/or fully open conformation, narrowly neutralizing antibodies can bind more efficiently to Env because the V3 loop, CD4-bs, and MPER are better exposed (14, 22, 26). It is thus possible that a virus could be relatively neutralization resistant if this equilibrium was shifted in favor of the more closed state. Supporting this idea, only the broadest and most potent neutralizing antibodies effectively bind to the virus in the closed spike conformation (12–14). Indeed, extensive screening of sera from HIV-1-infected individuals has resulted in the identification of relatively rarely elicited broadly neutralizing antibodies (bNAbs) that primarily target six epitopes on prefusion Env (1, 27): the V2-apex region (28, 29), the V3/glycan supersite (30–33), and the CD4-bs (34, 35) in gp120; the MPER (36–38) and, more recently, the fusion peptide (39, 40) in gp41; and the gp120-gp41 interface (41–43).

Here, we describe characteristics of the neutralization profile of a highly neutralization-resistant virus, 253-11 (44, 45), and explore the structure of its SOSIP trimer (46). Knowledge of the mechanisms used by HIV-1 to escape antibody neutralization has implications for immunogen design and for improving the efficacy of candidate HIV-1 vaccines that induce neutralizing antibodies.

RESULTS

Key sequence polymorphisms minimally explain 253-11 neutralization resistance.The 253-11 virus (44) is a tier 3 (47) subtype CRF02_AG virus that was previously shown to be highly resistant to neutralization by both clade-matched blood plasma (45) and subtype C sera (48). 253-11 is moderately to very resistant to MAb-mediated neutralization based upon data extracted from the Compile, Analyze, and Tally NAb Panels (CATNAP) tool (49) (data not shown). Here, we measured the sensitivity of 253-11 to different bNAbs in pseudovirus-based neutralization assays. The virus was resistant to neutralization by a range of bNAbs across several of the currently identified bNAb targets (Fig. 1A), even though the 253-11 amino acid sequence possesses most of the key residues that make up the different bNAb epitopes (Fig. 1B). 253-11 is strikingly resistant to most tested antibodies that target V3/glycans, despite possessing key potential N-linked glycosylation sites (PNGs), especially at positions 301 and 332 (30, 50), needed for the recognition of this class of antibodies (Fig. 1B). Notably, 253-11 possesses a 324GNIR327 sequence instead of the 324GDIR327 motif required for the binding of some N332-dependent antibodies (51, 52) (Fig. 1B). This might hinder the binding of many V3/glycan-targeted antibodies, although PGT124 has been shown to bind in the presence of N325 (51). To analyze the effect of this polymorphism, we created an N325D mutant of 253-11 and tested its sensitivity to PGT121, PGT126, PGT128, and PGT130, to which 253-11 is resistant. We found that the introduction of the 324GDIR327 motif made 253-11 modestly more sensitive to only one of the four antibodies tested (PGT121) (Fig. 1C), indicating that this critical sequence polymorphism in 253-11 is not a main contributor to the resistance of the virus to the panel of V3/glycan-specific bNAbs tested (Fig. 1A).

High neutralization resistance of 253-11 is due partially to the lack of bNAb epitopes and N-linked glycosylation. (A) Table showing the IC50s (micrograms per milliliter) obtained from pseudovirus-based neutralization of 253-11 by MAbs with different specificities. (B) Amino acid sequence of 253-11 (cytoplasmic domain not included) (GenBank accession no. ACC97453.1). Potential N-linked glycosylation sites (PNGs) are depicted in blue boxes. Key N-linked glycans involved in bNAb binding are numbered: N88 (fusion peptide); N156 and N160 (V2-apex); N276 (CD4 binding site); N301 and N332 (V3); and N88, N611, and N637 (gp120-gp41 interface). Residues forming other bNAb epitopes are annotated: loop D (which forms part of the CD4 binding epitope) (residues 273 to 283) (102), the CD4 binding loop (residues 364 to 373) (102), the MPER (membrane-proximal external region) (residues 660 to 683) (103), the fusion peptide (FP; residues 512 to 525) (40), the V1 loop (residues 131 to 155) (104), and the V2 loop (residues 158 to 196) (104). All numbering shown is according to the HXB2 sequence. (C) A 253-11 N325D mutant was engineered to recreate the V3 loop 324GDIR327 motif in the 253-11 virus. The sensitivities of the wild type (WT) and the N325D mutant to V3/glycan-specific antibodies were tested in pseudovirus-based neutralization assays, and the IC50 values (micrograms per milliliter) are shown. (D) Two PNGs, N293 and N363, present in 253-11 Env but not in 928-28 Env or COT6.15 Env, were removed by site-directed mutagenesis. Mutants were tested for sensitivity to serum samples from ART-naive, HIV-infected individuals that neutralized 253-11 WT poorly or not at all. For calculations, all serum-virus pairs in which the virus was resistant were considered to have an arbitrary ID50 value of 25. (E) Tier 2 and 3 viruses were previously tested for sensitivity to the 10 sera tested against the 253-11 N293A and N363A mutants (48). To rank the 253-11 N293A and N363A mutants within a panel of 24 viruses (48), geometric mean ID50 values were calculated by using all tested sera for each virus, and the viruses were ranked in order of neutralization resistance. Confidence intervals were calculated by using RStudio (84).

Because a longer V1/V2 loop has been associated with neutralization resistance (53, 54), we next analyzed the length of the 253-11 V1/V2 loop to determine whether it was unusually long. The V1/V2 loop of 253-11 is 65 amino acid residues long (Fig. 1B). Loop lengths of tier 3, tier 2, and tier 1 viruses are, on average, 72, 68, and 65 amino acids, respectively (49). Therefore, 253-11 does not have an unusually long V1/V2 loop but in fact resembles an average tier 1 virus in this regard, which suggests that loop length is not a key factor in the neutralization resistance phenotype of the virus.

The number of N-linked glycosylation sites (PNGs) on HIV-1 Env has also been found to be positively associated with neutralization resistance (55). 253-11 has 8 PNGs across the V1/V2 loops and 27 PNGs across gp120 overall (Table 1), numbers which are higher than the median values for tier 2 viruses, approximately 6 PNGs in V1/V2 and 25 PNGs in the entire trimer (55). To determine whether glycans play a role in the neutralization resistance of 253-11 by occluding key epitopes on the virus, we identified and mutated key potential PNGs that are found in 253-11 but not in a clade-matched neutralization-sensitive virus, 928-28, or in a well-characterized, neutralization-sensitive subtype C virus, COT6.15 (56) (Table 1). The other PNGs that are present in 253-11 but not in 928-28 and COT6.15 are in the highly variable V1 or V4 regions, and as such, it is difficult to determine which glycans are located at equivalent structural positions between viruses. Accordingly, we limited our mutational analysis of PNGs to positions 293 and 363 and tested the sensitivities of these mutants to sera that poorly neutralized (50% inhibitory dilution [ID50] of <100) or did not neutralize the wild-type (WT) 253-11 virus. The removal of either of the two glycans modestly increased the neutralization sensitivity of 253-11 to a small proportion of the 10 tested sera (Fig. 1D), probably by exposing underlying epitopes on Env (57). The sensitivities of 24 tier 2/3 viruses to these 10 serum samples were tested previously (48). There was no substantial change in the relative sensitivity of 253-11 upon the introduction of the N293A and N363A mutations (Fig. 1E). Since the neutralization resistance of 253-11 appears to be largely maintained in these glycosylation mutants, we propose that the neutralization resistance phenotype of 253-11 is only minimally due to shielding by these glycans.

Sequences outside the MPER control high resistance of 253-11 to MPER-specific neutralizing antibodies in sera.Pseudovirus-based neutralization assays revealed that 253-11 was sensitive to the most broadly neutralizing monoclonal antibodies (bNAbs) targeting the MPER (Fig. 1A). We further explored whether 253-11 was also sensitive to neutralization by the moderately broad and potent anti-MPER neutralizing antibodies commonly elicited in our cohort. We screened 217 unselected serum samples from a South African cohort of HIV-1-infected (>1 year) antiretroviral therapy (ART)-naive participants and found that WT 253-11 was resistant to or poorly neutralized by 91% of the sera (Fig. 2A).

Sensitivity of 253-11 to MPER-directed antibodies. (A) A total of 217 serum samples were tested for sensitivity to 253-11 and an HIV-2 isolate containing the 253-11 MPER sequence. The top panel is a schematic representation of the HIV-2/253MPER trimer constructs designed for this experiment. A threshold titer of 1:1,000 was used to define significant anti-MPER neutralization (48, 56), and a threshold titer of 1:100 was used to define substantial anti-253-11 neutralization. The HIV-2 backbone is derived from the 7312A virus. It was resistant to all but one of the 217 sera, which neutralized it with an ID50 of <300. (B) MPER sequences of clade-matched viruses 253-11 and 928-28 were swapped between the viruses by site-directed mutagenesis. A total of 10/19 sera from the upper left quadrant of panel A were tested for neutralization sensitivity to 253-11 and 928-28 MPER swap mutants. Neutralization was compared to that of the wild-type viruses for each mutant. (C) Two samples that did not neutralize WT 253-11 but exhibited high 253MPER-neutralizing activity were used for MPER antibody depletion using the MPR.03 peptide compared to a control scrambled-sequence peptide to determine whether MPER-specific antibodies were the major contributors to the neutralization of 928-28_253MPER mutants. SF162 was a negative control, and the HIV-2/HIV-1 chimeras with MPER sequences from 253-11, C1C, and C1 HIV-2/HIV-1 were the positive controls for depletion. Resistant sera are displayed throughout with an arbitrary ID50 value of 25.

To investigate the neutralization susceptibility of the 253-11 MPER, we constructed a chimeric virus containing an HIV-2 backbone with the 253-11 MPER sequence (HIV-2/253MPER) (Fig. 2A). Only two sera (Fig. 2A, upper right quadrant) substantially neutralized both native 253-11 virus and HIV-2 displaying a 253-11 MPER, and only one of these serum samples exhibited a drop in neutralization upon the depletion of anti-MPER antibodies (data not shown). This further indicates that 253-11 is unusually resistant to commonly elicited serum anti-MPER antibodies.

Interestingly, 19 sera (8.8%) recognized the 253-11 MPER in the HIV-2/253MPER chimeric construct but poorly neutralized the WT 253-11 virus (ID50 of <100) (Fig. 2A, upper left quadrant). A threshold of an ID50 of >1,000 was chosen as an identifier of anti-MPER antibodies in sera (48, 56, 58–60). Six of these 19 sera were tested and shown to neutralize other tier 2/3 HIV-1 isolates by specifically targeting the MPER (48), which further suggests that 253-11 is unusually resistant to anti-MPER antibodies in unselected sera, compared to other HIV-1 isolates.

To determine whether the resistance of WT 253-11 to anti-MPER antibodies in sera was induced by polymorphisms in the MPER itself, we exchanged MPER amino acid sequences between 253-11 and 928-28, the clade-matched virus that is substantially less neutralization resistant (47, 48). We tested these MPER swaps for sensitivity to 10/19 sera from the upper left quadrant (Fig. 2A) and observed that the pattern of neutralization generally followed the Env “backbone” and not the MPER sequence (Fig. 2B), suggesting that sequence differences outside the MPER itself were the cause of 253-11 MPER neutralization resistance. We previously confirmed, using anti-MPER depletion experiments, that the primary target of neutralizing antibodies was the MPER, even for the 253-11 chimera displaying the 928-28 MPER (48). Two sera tested in the same manner with the 928-28 chimera containing the 253MPER sequence (928-28_253MPER) also neutralized virus primarily via the recognition of the MPER (Fig. 2C). Combined, our data thus strongly suggest that 253-11 does not evade neutralization by commonly elicited MPER-specific antibodies through sequence polymorphisms within the MPER but rather does so through presumably structural Env features outside the MPER.

Impact of MPER mutations on sensitivity of 253-11 to bNAbs.We next studied the effects of MPER mutations L669S and Y681H on the neutralization resistance of 253-11. The L669S and Y681H mutations have been described as increasing the lifetime of the “open” HIV-1 Env conformation after CD4 has bound by decreasing the rate at which cell fusion occurs (25, 61). These mutations are made at two positions that are almost completely invariant in native sequences (49). Previous studies found that the introduction of these MPER mutations results in increased sensitivity to certain MAbs that have epitopes preferentially exposed in the post-CD4 open conformation (25, 61). We used these mutations as a probe to better understand the mechanism of the high neutralization resistance of 253-11. Although direct effects of these amino acid changes on the MPER structure cannot be ruled out, the strongest effect of these mutations that has been proposed previously is altering larger aspects of structure and function (25, 61).

We tested the L669S and Y681H mutants of 253-11 for sensitivity to bNAbs that target five of the major sites of vulnerability on HIV-1 Env. We observed that sensitivity to CAP256.VRC26.25 (targeting the V2-apex), VRC03 (targeting the CD4-bs), and 10-1074 and PGT128 (targeting the V3/glycans) was increased for at least one mutant of 253-11 (Fig. 3). Several bNAbs that target other sites were not affected by the MPER mutants, including PGT151, targeting the gp120-gp41 interface, and VRC01, NIH45-46 G54W (referred to as NIH45-46), and 3BNC117, targeting the CD4-bs (Fig. 3). There were a few increases in the sensitivities of the MPER mutants to bNAbs targeting the MPER, primarily 4E10, 2F5, Z13e1, and 10E8, and to soluble CD4 (sCD4). As expected, 928-28 MPER mutants displayed some of the same changes in antibody sensitivity as the 253-11 MPER mutants (Fig. 3). Previously, the primary effect of the L669S and Y681H mutations had been proposed to prolong the lifetime of the post-CD4 open conformation (25, 61). Strikingly, these mutations increase the neutralization of 253-11 by antibodies that are known to preferentially bind the closed trimer spike conformation of HIV-1 Env: 10-1074 (62), PGT128 (63), and CAP256.VRC26.25 (64).

Impact of L669S and Y681H mutants on bNAb recognition. Values for the neutralization of WT 253-11, WT 928-28, and their L669S/Y681H mutants by MAbs targeting most of the broadly neutralizing antibody epitopes are displayed in micrograms per milliliter. For fold difference calculations, resistant combinations (IC50 of >20 μg/ml) were assigned an arbitrary value of 40 μg/ml.

Surprisingly, the MPER mutants decreased the sensitivity of 253-11 to bNAb PG9 approximately 10-fold (Fig. 3). PG9 binds to a conformation-dependent epitope in the V2-apex that preferentially exists in the pre-CD4-bound Env spike (29). We hypothesize that the decrease in neutralization may be due to a destabilization of the PG9 quaternary epitope at the apex of the prefusion trimer or to differential glycan processing as a result of the mutations, as PG9 is dependent on strict glycoforms for binding (28, 29, 65). This effect was seen only in 253-11; no change in sensitivity to PG9 was observed when the same mutations were introduced into 928-28 (Fig. 3).

MPER mutations alter the exposure of 253-11 Env epitopes to neutralizing antibodies from sera.We tested the 253-11 MPER mutants for sensitivity to 12/19 sera that neutralize the 253-11 MPER in the chimeric virus but not in the WT (Fig. 2A, upper left quadrant). In all cases, the L669S and Y681H mutations increased the sensitivity of 253-11 to these sera, with L669S frequently having a greater effect than Y681H (Fig. 4A).

253-11 L669S and Y681H mutants generally have increased sensitivity compared to WT 253-11. (A) Neutralization of WT 253-11 and the L669S/Y681H mutants by 12/19 of the serum samples that recognize 253MPER in the HIV-2/HIV-1 MPER chimera but not in the WT 253-11 virus was assessed. For fold difference calculations, resistant combinations (ID50 of <50) were assigned an arbitrary ID50 value of 25. (B) Two serum samples that neutralized 253-11 poorly (ID50 of <100) but exhibited high 253MPER-neutralizing activity were used for MPER antibody depletion using the MPR.03 peptide compared to a control scrambled-sequence peptide to determine whether MPER-specific antibodies were the major contributors to the increase in neutralization observed with the L669S and Y681H mutant viruses. SF162 was a negative control for depletion, and the HIV-2/HIV-1 chimeras with MPER sequences from 253-11, C1C, and C1 HIV-2/HIV-1 were the positive controls.

To illustrate that the L669S and Y681H mutations were directly affecting the recognition of not only the 253-11 MPER-specific antibodies in these samples but also other epitopes, we depleted MPER antibodies from 2 samples from the 19 sera that have dominant neutralizing anti-MPER antibodies. Depletion was confirmed by testing the capacity to neutralize three HIV-2/HIV-1 MPER chimeras, with reductions of 40- to 115-fold being observed (Fig. 4B). When the 253-11 L669S and Y681H mutants were tested for sensitivity to these MPER-depleted samples, we observed substantial residual neutralization, particularly for the L669S mutant, suggesting that antibodies targeting epitopes other than the MPER were also responsible for the increase in the neutralization sensitivity of the L669S and Y681H mutants (Fig. 4B). Together, our data suggest that the global neutralization-resistant phenotype of tier 3 viruses can be shifted by these single point mutations, plausibly to tier 1 or 2 phenotypes, through changes in the recognition of several epitopes, including epitopes thought to be recognized primarily in the pre-CD4-bound trimer conformation.

253-11 SOSIP trimers are stable and predominantly adopt a prefusion conformation.To probe the ability of the 253-11 sequence to preferentially adopt a prefusion Env conformation, we engineered a 253-11 SOSIP Env trimer, as previously described (46). 253-11 SOSIP trimers eluted at the same volume (~10 ml) by size exclusion chromatography (SEC) as the gold-standard BG505 SOSIP trimers (Fig. 5A). 253-11 SOSIP trimers were expressed in slightly higher yields than those of BG505 SOSIP trimers and were efficiently cleaved into gp120/gp41 subunits, as observed by SDS-PAGE under reducing conditions (Fig. 5A). Differential scanning fluorimetry revealed that the 253-11 SOSIP trimers have a melting temperature (Tm) (62.2°C) similar to that of BG505 SOSIP trimers (65.8°C) (Fig. 5B) and are comparable to the most thermostable SOSIP.664 trimers that have been described to date (66). We also note a more pronounced pH dependence for the thermostability of 253-11 SOSIP trimers than for BG505 SOSIP trimers (Fig. 5C). The antigenicity of 253-11 SOSIP trimers was assessed by measuring the binding of three bNAbs, VRC01, PG9, and 10-1074 Fabs, and two nonneutralizing MAbs, 17b and F240, to the trimer. As Fabs, VRC01, 10-1074, and PG9 bound the trimer with KD (equilibrium dissociation constant) values of 11.8, 25.0, and 137 nM, respectively (Fig. 5D and E), suggesting that 253-11 SOSIP trimers are well folded and correctly display conformational epitopes. 253-11 SOSIP trimers were not bound by MAbs 17b and F240 (Fig. 5D and E), which suggests that these trimers efficiently mask these nonneutralizing epitopes (Fig. 1A) in the prefusion conformation.

Biophysical characterization of 253-11 SOSIP trimers. (A) Size exclusion chromatography of HIV-1 Env SOSIP trimers of 253-11 (blue curve) and BG505 (red curve) sequences with detection shown in milliabsorption units (mAU). Shown are SDS-PAGE gels of purified 253-11 and BG505 SOSIP trimers under nonreducing (NR) and reducing (R) conditions. In the 253-11 gel, two sections of a single gel were spliced together for bands to be displayed next to the protein ladder. (B) Tm of 253-11 and BG505 SOSIP trimers in buffer containing 50 mM Tris (pH 9.0) and 150 mM NaCl using differential scanning fluorimetry. Shown are fluorescence emission spectra for 253-11 and BG505 SOSIP trimers (in black) with the corresponding differential of the curve (in gray). Tm values are marked with a line and calculated as the temperature at which there is an equal population of folded and unfolded proteins in solution. Unfolding is measured by changes in the barycentric mean (BCM) fluorescence wavelength, measured in nanometers. (C) Tm of 253-11 and BG505 SOSIP trimers measured in 50 mM sodium acetate (pH 5.6), 50 mM HEPES (pH 7), and 50 mM Tris (pH 9). Error bars are shown and represent the standard deviations from the means derived from two independent differential scanning fluorimetry experiments. (D) Representative biolayer interferometry curves showing that the VRC01, PG9, and 10-1074 Fabs bind to 253-11 SOSIP.664 trimers and that antibodies 17b and F240 do not. (E) Comparison of calculated IC50 values (micrograms per milliliter) from neutralization assays and KD values (nanomolar) obtained from BLI for binding of the VRC01, PG9, and 10-1074 Fabs to 253-11 SOSIP trimers. The right panel shows the key. NB, no binding.

Small-angle X-ray scattering (SAXS) measurements coupled to size exclusion chromatography showed that the 253-11 SOSIP trimers were almost entirely free of aggregation and were properly folded (Fig. 6 and Table 2). The calculated diameter (Dmax) for the 253-11 SOSIP trimers (152.4 Å) is comparable to that for the BG505 SOSIP trimers (146.2 Å) (Table 2 and Fig. 6A). Although our 253-11 and BG505 SOSIP constructs analyzed by SAXS contain a His6 tag, the Dmax of BG505 SOSIP trimers is almost identical to that of BG505 SOSIP trimers without the tag, as previously reported (147 Å) (67). The low-resolution three-dimensional volume obtained from SAXS measurements further demonstrated that 253-11 SOSIP trimers adopt a closed conformation with dimensions similar to those of the BG505 SOSIP trimers (Fig. 6A).

Crystal structure of the 253-11 trimer reveals attributes of gp41 and gp120 compactness.To gain a further understanding of whether 253-11 Env possesses structural differences from other HIV-1 Env proteins that might explain its neutralization-resistant phenotype, we crystallized the 253-11 SOSIP trimer in complex with the 10-1074 Fab and solved its structure at a 6.5-Å resolution (Table 3). As expected, the 253-11 SOSIP trimer adopts a closed, prefusion conformation, with three 10-1074 Fabs protruding from the membrane-distal gp120 subunits, contacting glycans at the base of the V3 loop (Fig. 7A). Electron density maps revealed distinct densities for gp41 heptad repeat 1 (HR1) and HR2 helices (Fig. 7B and C). Compared to the BG505 SOSIP trimer that was used as the search model for molecular replacement (Protein Data Bank [PDB] accession no. 5T3X), the membrane-proximal portion of HR1 helices are shifted inwards toward the trimer axis in the 253-11 SOSIP trimer crystal structure (Fig. 7B). We note that HR2 helices in gp41 are also displaced toward the trimer axis in the 253-11 SOSIP trimer crystal structure (Fig. 7C); however, as was reported previously (68–70), this region is flexible in SOSIP constructs terminating at position 664 and is involved in crystal packing here. As such, we cannot conclude whether this observation highlights a specific attribute of 253-11 Env or, more likely, is induced by the arrangement of 253-11 SOSIP trimers in the crystal lattice. As an internal reference, such differences in the dispositions of helices between 253-11 and the BG505 SOSIP trimers were not observed in gp120; all structural elements aligned well in this subunit (e.g., gp120 α1) (Fig. 7D). Comparison of the crystal structure of the 253-11 SOSIP trimer with other HIV-1 trimer structures from BG505, 16055, X1193.c1, and JR-FL in complex with various antibodies (PDB accession no. 5T3X, 5CEZ, 5FYK, 5FYJ, 4ZMJ, 5ACO, 5C7K, 5FYL, 5I8H, 5D9Q, 5THR, 5U1F, 5UTY, 5UTF, 5V8L, 5V8M, and 5UM8) revealed that the 253-11 SOSIP trimer in complex with the 10-1074 Fab has HR helices that are more tightly positioned toward the trimer axis than for all other solved HIV-1 trimer structures, except for BG505 SOSIP trimers in complex with the PGT128 and 8ANC195 Fabs (PDB accession no. 5C7K) (Fig. 7E).

Crystal structure of the 253-11 SOSIP trimer in complex with the 10-1074 Fab. (A) Side view of the crystal structure of the 253-11 SOSIP trimer in complex with the 10-1074 Fab. One of the protomers is highlighted. gp120 and gp41 are shown in yellow and cyan, respectively, and represented as a surface and a cartoon. The heavy chain (in black) and the light chain (in gray) of the 10-1074 Fab are represented as a cartoon and are binding to the V3 base, specifically to the N332 glycan (represented as brown spheres). N-linked glycans for gp120 and gp41 are represented as spheres in yellow and cyan, respectively. (B and C) Bottom view of the 253-11 SOSIP trimer crystal structure showing the Fo − Fc electron density map (green mesh) obtained after molecular replacement for HR1 (B) and HR2 (C). (D) Bottom view of the 253-11 SOSIP trimer showing the 2Fo − Fc electron density map (blue mesh) for the gp120 α1 helices. (E) One 253-11 SOSIP protomer represented as a cartoon, with gp120 (yellow) and gp41 (cyan) represented along the trimer axis (dashed black line). 253-11 is superimposed with the HR1 and HR2 helices from BG505 (PDB accession no. 5T3X, 5CEZ, 4ZMJ, 5ACO, 5C7K, 5FYL, 5I8H, 5D9Q, 5THR, 5U1F, 5UTY, 5UTF, 5V8L, and 5V8M), JR-FL (PDB accession no. 5FYK), X1193.c1 (PDB accession no. 5FYJ), and 16055 (PDB accession no. 5UM8) trimers (all shown as cartoons in gray).

After molecular replacement, we also noted a clear difference in the disposition of 253-11 gp120 protomers along the trimer axis. Rigid-body refinement slightly repositioned the gp120 protomers in closer proximity to the trimer axis. To investigate how gp120 protomers arranged themselves within the 253-11 SOSIP trimer compared to trimers of other sequences, we calculated distances between the centers of mass of each of the three 253-11 gp120 protomers and compared the interprotomer distances with those for other trimers for which the structures have been solved to date by X-ray crystallography and cryo-electron microscopy (cryo-EM) (Fig. 8). Specifically, we compared gp120 interprotomer distances for 253-11 and the ligand-free BG505 trimer (PDB accession no. 4ZMJ); BG505 in complex with Fabs (PDB accession no. 5T3X, 5CEZ, 4NCO, 3J5M, 4TVP, 5ACO, 5C7K, 5FYL, 5I8H, 5D9Q, 5THR, 5U1F, 5JSA, 5JS9, 5UTY, 5UTF, 5V8L, 5V8M, and 5UM8); and trimers derived from JR-FL (clade B) (PDB accession no. 5FYK), X1193.c1 (clade G) (PBD accession no. 5FYJ), and 16055 (clade C) (PDB accession no. 5UM8) isolates (Fig. 8). 253-11 had smaller gp120 interprotomer distances than those of the clade B, C, and G SOSIP trimers and gp120 interprotomer distances that were smaller than or comparable to those of 13 of the 19 BG505 structures (PDB accession no. 5ACO, 5CEZ, 5JSA, 5JS9, 5C7K, 3J5M, 5U1F, 4TVP, 4NCO, 5T3X, 5V8L, and 5V8M). As an internal validation of our analysis, the CD4/17b/8ANC195-bound structure (open trimer) of BG505 (PDB accession no. 5THR) had the highest interprotomer distances out of the trimers analyzed (Fig. 8C). Notably, our analysis revealed that engineered stabilizations in BG505 trimers (71, 72) (PDB accession no. 5U1F, 5UTY, and 5UTF) did not substantially affect the distances between the three gp120 protomers. Taken together, our structural data suggest that the 253-11 SOSIP trimer has subtle differences compared to the JR-FL, 16055, and X1193.c1 SOSIP structures and some of the BG505 trimer structures. We postulate that these differences, albeit observed at low resolution, might contribute to the neutralization resistance of 253-11.

gp120 compactness of the 253-11 SOSIP trimer. (A) Top view of the 253-11 SOSIP trimer showing the three gp120 (yellow) protomers. The center of mass (blue spheres) for each gp120 protomer was calculated using PyMOL (73). The calculated distances between protomers are represented as a blue line with the corresponding values. (B) Superposition of the center of mass for each of the gp120 protomers for the 253-11 SOSIP trimer (blue spheres) with 19 other trimers. Green, BG505 open trimer (PDB accession no. 5THR); yellow, X1193.c1; orange, JR-FL (PDB accession no. 5FYK); pink, 16055 (PBD accession no. 5UM8); gray, BG505 (PBD accession no. 4ZMJ, 5T3X, 5CEZ, 4NCO, 3J5M, 4TVP, 5ACO, 5C7K, 5FYL, 5I8H, 5D9Q, 5U1F, 5JSA, 5JS9, 5UTY, 5UTF, 5V8L, and 5V8M). (C) Individual gp120 interprotomer distances are shown for the trimers ranked from the smallest to the largest. The 253-11 SOSIP trimer is highlighted in blue.

DISCUSSION

A global HIV-1 vaccine will likely need to protect against neutralization-resistant viruses to effectively reduce the incidence of HIV-1. Therefore, an understanding of the Env structure of neutralization-resistant viruses is critical. Most HIV-1 immunogen research focuses on the closed conformation of Env to elicit bNAbs; however, immunization studies with BG505 and other tier 2 SOSIP trimers have yielded primarily autologous responses (73–75), despite further modifications to increase trimer stability (71, 74) and high-efficiency delivery methods, such as nanoparticle presentation (76). Therefore, a further understanding of the mechanisms used by tier 3 HIV-1 isolates to evade neutralizing responses and the development of trimers based on these tier 3 viruses may aid in the design of immunogens capable of eliciting antibodies that have higher strain coverage. In this report, we studied a highly neutralization-resistant tier 3 subtype CRF02_AG virus obtained from Cameroon, 253-11 (44). We present evidence that 253-11 is particularly resistant to antibody neutralization only minimally because of key sequence polymorphisms and changes in its glycan shield. Although we were not able to ascertain a structural basis for its very high neutralization resistance, we noted its highly compact trimer spike at low resolution, which was more compact than Env structures solved for JR-FL (clade B) (PDB accession no. 5FYK), X1193.c1 (clade G) (PDB accession no. 5FYJ), and 16055 (clade C) (PDB accession no. 5UM8). The 253-11 SOSIP was smaller than some, but not all, BG505 Env structures. This is despite the fact that the 253-11 virus (tier 3) is substantially more neutralization resistant than BG505 (tier 2).

The introduction of the L669S (25) and Y681H (61) mutations into the MPER of viruses was previously shown to shift virus entry kinetics by delaying fusion and, thus, increase the neutralization sensitivity of viruses. These mutations affected the sensitivity of the 253-11 virus to bNAbs targeting the V2-apex (N160 dependent), the V3/glycans, and the MPER and also included increased sensitivity to sCD4. Strikingly, the mutations decreased the neutralization sensitivity of 253-11 to PG9, a conformation-dependent antibody targeting the V2-apex in the Env trimer spike, suggesting a disruption of the PG9 epitope possibly through the destabilization of the closed Env trimer structure of 253-11. The mutations did not alter the sensitivity of clade-matched 928-28 to PG9, suggesting an effect relatively specific for 253-11. The WT virus and the L669S and Y681H mutants were resistant to both PGT145 and PG16; i.e., the presence of these mutations did not result in changes in sensitivity to these two bNAbs. This is consistent with the possibility that their specific epitopes are not present in 253-11. The L669S and Y681H mutants of 253-11 were more sensitive to CAP256.VRC26.25; however, there are discrete differences between the epitopes for CAP256.VRC26.25 and PG9, most notably that the former is not dependent on the N160 glycan (64). Thus, it is plausible that while the epitope for PG9 was altered by the mutations, the epitope for CAP256.VRC26.25 merely became more exposed.

The effects of the L669S and Y681H mutations suggest a change in the exposure of a range of epitopes across Env, including the V2-apex and the V3/glycans, which are known to be preferentially exposed in the pre-CD4 conformation (62, 63, 77, 78). The single-point mutations dramatically shifted the tier phenotypes of 253-11 and other viruses to which they had been added (25, 61).

In order to gain direct insight into the structure of 253-11 Env, we constructed and crystallized a 253-11 SOSIP Env trimer. The crystal structure of the 253-11 trimer in complex with the 10-1074 Fab indicated that the gp41 HR1 and HR2 helices are in a more compact disposition relative to the trimer axis than in JR-FL, X1193.c1, and 16055 trimer structures and some BG505 trimer structures that have been solved to date. In addition, our data support a molecular mechanism of interconnectedness between inward movements in gp41 HR helices and relatively small distances between gp120 protomers for 253-11. Together, these data suggest a compact phenotype that coexists with the high neutralization resistance of this virus. We hypothesize that the 253-11 MPER may be less accessible because of the disposition of gp41 HR helices. This may explain why 253-11 is so resistant to commonly elicited anti-MPER antibodies that are prevalent in sera. However, we cannot address this point directly because the MPER is not present in the SOSIP.664 constructs that we used.

It seems plausible that the equilibrium of compact Env spikes, such as those of 253-11, more strongly favors a closed conformation that is less likely to enter transient open conformations than spikes of most other HIV-1 isolates (14, 79). To date, few SOSIP trimers based on highly neutralization-resistant viruses have been reported in the literature. One example is PVO.4 (80), which is more neutralization sensitive than 253-11 when tested for sensitivity to a panel of subtype C sera (48) and subtype CRF02_AG blood plasma samples (45). The development of a SOSIP trimer that contains the neutralization resistance and Env compactness properties of the 253-11 virus thus enables future studies of tier 3 Env immunogenicity.

Taken together, our results show the molecular characteristics of a tier 3 virus. We show that 253-11 is extremely resistant to neutralization by commonly elicited, moderately neutralizing antibodies and is sensitive to only some of the most broadly neutralizing, more rarely elicited antibodies. These findings suggest that some tier 3 viruses that resemble 253-11 might be able to circulate in a population of vaccinated individuals who have only moderately broad neutralizing antibody responses. It is therefore important to develop a vaccine capable of protecting against all or most HIV-1 strains, including the most neutralization-resistant tier 3 viruses.

MATERIALS AND METHODS

Serum samples and monoclonal antibodies.All serum samples used in this study were collected from study participants who were recruited from (i) caregivers of patients at the pediatric HIV clinic at Groote Schuur Hospital (n = 92) and (ii) attendees of the HIV wellness clinic at the Khayelitsha Site B clinic (n = 125). Both clinics are in Cape Town, South Africa. In all, 217 blood samples were collected between December 2009 and July 2011 from donors who were >18 years old, chronically HIV-1 infected (>1 year), and not exposed to ART, except for ART given for the prevention of mother-to-child transmission (>3 months prior to sample collection). The median CD4+ T cell count of the donors was 425 (interquartile range [IQR], 305, 545). Viral loads were measured for 50 of the 217 samples; the median viral load was 27,000 (IQR, 8,150, 100,000). MAbs 2F5, PG9, PG16, and Z13e1 were obtained from Polymun Scientific, and MAbs PGT128, PGT130, PGT151, CAP256.VRC26.25, and F105 were kind gifts from P. Moore, National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. All other MAbs were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID.

Pseudovirus constructs.The pSG3Δenv HIV-1 backbone was obtained via the NIH AIDS Reagent Program from John C. Kappes and Xiaoyun Wu. Cloned HIV-1 envelope constructs, including 253-11 (44) (GenBank accession no. EU513191.1), were obtained through the NIH AIDS Reagent Program. The 7312A parent HIV-2 genomic clone and the HIV-2 C1 (105) and C1C chimeric constructs (60) were kindly provided by George Shaw, University of Pennsylvania, Philadelphia, PA. The HIV-2/253MPER chimeric construct was produced by mutagenesis from HIV-2 chimeric construct C1 by using QuikChange II XL site-directed mutagenesis kit 121 (Stratagene). L669S (81) and Y681H (61) mutants of 253-11 and 928-28 as well as 253-11_928MPER and 928-28_253MPER swap mutants were generated by using the GeneArt site-directed mutagenesis plus kit (Invitrogen). All intended mutations and the absence of unrelated PCR errors were confirmed by sequencing of both strands of the entire env open reading frame.

Generation of pseudoviruses and neutralization assays.Pseudoviruses were prepared and tested as previously described (82). Briefly, env DNA was cotransfected with pSG3Δenv (83) into human embryonic kidney HEK293T cells (obtained from Andrew Rice via the NIH AIDS Reagent Program). The pseudovirus-containing supernatant was harvested at 48 h posttransfection and stored in single-use aliquots at −80°C. Neutralization was tested by using a standard pseudovirus-based neutralization assay. Antibody and virus were incubated for 1 h at 37°C. TZM-bl cells (obtained from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc., via the NIH AIDS Reagent Program) at 104 cells/well were added to the antibody-virus combination and incubated at 37°C for 48 h. Titers (dilution of serum that inhibits 50% of infection [ID50] or concentration of MAb that inhibits 50% of infection [50% inhibitory concentration {IC50}]) were calculated by using curve fit functions in Prism (GraphPad). Sera were tested starting at a 1:50 dilution with further serial 2-fold dilutions. To rank the 253-11 N293A and N363A mutants within a panel of 24 viruses (48), geometric mean ID50 values were calculated by using all tested sera for each virus, and the viruses were ranked in order of neutralization resistance. Confidence intervals for each geometric mean ID50 value were calculated by using RStudio (84). On log scale axes, undetectable neutralization was plotted with arbitrary values of an ID50 of 25.

Depletion of MPER-specific antibodies.Sera were tested for their capacity to neutralize HIV-1 isolates or chimeric HIV-2 constructs by the recognition of the MPER. Antibodies were depleted in two rounds of depletion, as previously described (48, 56, 85), using a biotinylated MPER peptide (MPR.03 [KKKNEQELLELDKWASLWNWFDITNWLWYIRKKK-biotin-NH2]; Peptide Synthetics) (86). The completeness of depletion was tested by measuring the reduction of neutralizing activity toward HIV-2/HIV-1 MPER chimeras, and neutralization-sensitive HIV-1 isolate SF162 was used as a negative control because we expect antibodies other than anti-MPER antibodies to dominate the neutralization of this virus (87). Control depletions were performed as described above, using a biotinylated control peptide with a scrambled sequence, KKKNEKSNNDWERLWLEWLYIWLQDWAFTLIKKK-biotin-NH2. A threshold of a ≥3-fold drop in the ID50 compared to control peptide depletion was accepted as positive for MPER-mediated neutralization.

253-11 SOSIP, BG505 SOSIP, VRC01, PG9, and 10-1074 construct design.All sequences were codon optimized for expression in human cells and synthesized by GeneArt (Life Technologies). 253-11 and BG505 SOSIP.664 constructs were subcloned into the pHLsec vector (88), using the restriction enzymes AgeI and KpnI, such that a His6 tag was located at the C terminus of the construct to facilitate affinity purification. The addition of the stop codon before the His6 tag allowed us to remove the tag and produce an untagged 253-11 trimer for crystallization. The heavy chains and light chains of the 10-1074, PG9, and VRC01 Fabs were subcloned into the pHLsec vector (88), using the restriction enzymes AgeI and KpnI.

Expression and purification of 253-11 and BG505 SOSIP trimers.253-11 SOSIP.664 and BG505 SOSIP.664 plasmids were transiently cotransfected with a furin protease plasmid into HEK293F (Thermo Fisher Scientific) or HEK293 Gnt I−/− (HEK293S) (ATCC CRL-3022) cells in suspension. Cells were split in 200-ml cultures at 0.8 × 106 cells/ml. Fifty micrograms of DNA was filtered and mixed in a 1:1 ratio with FectoPro transfection reagent (Polyplustransfection). The DNA-FectoPro solution was incubated with the cells at 37°C for 6 to 7 days before harvesting. Cells were harvested by centrifugation, and supernatants were filtered by using a 0.22-μm Steritop filter (EMD Millipore). The supernatant containing the protein with a His6 tag was passed through a HisTrap Ni-nitrilotriacetic acid (NTA) column (GE Healthcare). The column was washed with buffer containing 1× phosphate-buffered saline (PBS) (pH 7.4) and 5 mM imidazole prior to elution with an increasing gradient of imidazole up to 500 mM. The untagged 253-11 trimer protein was purified through a Galanthus nivalis lectin column. The trimer was washed with 1× PBS and then with 0.5 M sodium chloride (NaCl) in 1× PBS, followed by an additional wash with 1× PBS. The trimer was eluted with 1 M methyl-α-d-mannopyranoside (MMP) in 1× PBS. In both cases, the affinity-purified proteins were further purified to size homogeneity by using two runs of Superdex 200 Increase 10/300 GL size exclusion chromatography (GE Healthcare) in buffer containing 20 mM Tris (pH 9.0) and 150 mM NaCl. The purity of SOSIP.664 trimers was assessed by using nonreducing and reducing SDS-PAGE gels stained with Quick Coomassie blue (Generon) for visualization.

Expression and purification of 10-1074, VRC01, and PG9 Fabs.HEK293F cells were transfected with heavy and light chain plasmids (2:1 ratio) with a total DNA amount of 90 μg for each 200-ml culture. FectoPro (Polyplus Transfection) was used as a transfection reagent in a 1:1 ratio of DNA to FectoPro. Cells were transfected at a cell density of 0.8 × 106 cells/ml and incubated at 37°C for 6 to 7 days. Cells were harvested, and supernatants were retained and filtered through a 0.22-μm membrane. Supernatants were flowed through an anti-Kappa affinity column (GE Healthcare) or an anti-Lambda affinity column (GE Healthcare) by using an Akta Start chromatography system (GE Healthcare) and eluted with 100 mM glycine (pH 2.2 to 2.7). Eluted fractions were immediately neutralized with 1 M Tris-HCl (pH 9.0). Fractions containing protein were buffer exchanged into 20 mM sodium acetate (pH 5.6). Ion exchange chromatography was performed by using a MonoS column (GE Healthcare) and eluted with a potassium chloride gradient. Fractions were pooled, concentrated, and flowed through a Superdex 200 Increase 10/300 GL column (GE Healthcare) in 20 mM sodium acetate (pH 5.6) to obtain a purified sample. The purity of protein samples was assessed by using nonreducing and reducing SDS-PAGE gels stained with Quick Coomassie blue (Generon) for visualization.

SAXS data collection and processing and structure determination.253-11 and BG505 SOSIP.664 trimers expressed in HEK293F cells were subjected to SAXS measurements with a Superdex-200 size exclusion column (SEC-SAXS) at a flow rate of 0.7 ml/min at the BioSAXS 18-ID-D beamline at the Argonne Photon Source (Chicago, IL, USA). Images were collected after a 1-s exposure. Buffer control samples were derived from regions in the SEC-SAXS profile preceding the elution of the sample and were used to correct the scattering curves. Approximately 10 frames from the SEC peak were averaged to generate an idealized scattering curve by using PRIMUS (89). Analysis of the scattering curve by using PRIMUS showed no signs of aggregation, long-range interactions, or radiation damage. Scattering data were processed until the q value was equal to 0.2 Å−1. Determinations of Dmax and overall shape were performed with high confidence via analysis of the P(r) function, Kratky plots [I(q)q2 versus q], and the Porod invariant. Ten ab initio models were generated by using DAMMIF (90), imposing P3 symmetry. Ab initio models were aligned and averaged by using DAMAVER (91) and DAMFILT (91) to yield a final low-resolution model. The high quality of the χ2 values for all DAMMIF models is shown in Table 2. Chimera was used to visualize and dock the atomic structures into the SAXS envelopes. The quality of the model was further analyzed with CRYSOL (92), using the crystal structures of 253-11 and BG505 SOSIP.664 trimers (PDB accession no. 5T3X).

Determination of the thermostability of 253-11 and BG505 SOSIP trimers.Conformational stability was assessed by measuring the Tm monitored by an intrinsic fluorescence intensity ratio (350 nm/330 nm) using a UNit instrument (Unchained Labs). SEC-purified SOSIP trimer samples were concentrated to 1 mg/ml and heated from 20°C to 90°C in 1°C increments, with an equilibration time of 60 s before each measurement. Trimer thermostability was measured in three different buffers: 50 mM Tris (pH 9), 50 mM HEPES (pH 7), and 50 mM sodium acetate (pH 5.6). All buffers contained 150 mM NaCl. Data were processed using the standard UNit analysis software. Sample measurements were made in duplicate, values were averaged, and standard errors were calculated.

Fab and antibody binding assay using biolayer interferometry.The binding affinities of the VRC01, PG9, and 10-1074 Fabs and the 17b and F240 antibodies for the 253-11 SOSIP trimer were measured by biolayer interferometry (BLI) using the Octet Red system (Pall FortéBio). Fab or antibody binding biosensors were hydrated in 1× kinetics buffer (1× PBS [pH 7.4], 0.002% Tween, 0.01% bovine serum albumin [BSA]) and loaded with 10 μg/ml Fab/antibody for 60 s. Biosensors were then transferred into wells containing 1× kinetics buffer to achieve a baseline value before being transferred into wells containing serial dilutions of the 253-11 SOSIP.664 trimer starting at 1,500 nM and decreasing to 187.5 nM. The association phase was followed by a dissociation step with 1× kinetics buffer. Analysis was performed using the Octet software, with a 1:1 fit model. Experiments were repeated in triplicates, and values were averaged.

Crystallization and structural determination of the 253-11 SOSIP trimer in complex with the 10-1074 Fab.A molar excess of the 10-1074 Fab was added to the purified 253-11 SOSIP trimer expressed in HEK293 GnT I−/− cells. To obtain deglycosylated samples, the complex was treated with the enzyme endoglycosidase H (New England BioLabs) for 1 h at 37°C. The deglycosylated 253-11 SOSIP trimer in complex with the 10-1074 Fab was purified by size exclusion chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare). The purified 253-11 SOSIP trimer–10-1074 Fab complex was concentrated to 5 mg/ml in a buffer containing 20 mM Tris (pH 9.0) and 150 mM NaCl. Initial crystals were obtained by hanging-drop vapor diffusion in a solution containing 0.5 M sodium chloride, 1.9 M ammonium sulfate, and 0.1 M sodium cacodylate (pH 6.5). Crystals were improved by microseeding after mixing 0.6 μl protein, 0.4 μl solution, and 0.2 μl crystal seeds. Crystals were cryoprotected by soaking in a mother liquor solution containing 20% glycerol and flash cooled in liquid nitrogen. X-ray diffraction data were collected at the 23-ID-D beamline at the Argonne Photon Source (Chicago, IL, USA). A unique data set for the 253-11 SOSIP trimer–10-1074 Fab complex was processed by using XDS (93). Based on the R3:H space group and Matthews volume calculation (94–96), we estimated that there were two molecules of the 253-11 SOSIP protomer–10-1074 Fab complex in the asymmetric unit. The crystal structure was solved by molecular replacement using one protomer of the BG505 SOSIP in complex with the 10-1074 Fab (PDB accession no. 5T3X) as a search model in Phaser (97). The structure was refined by manual building in Coot (98) and by using phenix.refine (99) with strict reference model and secondary-structure restraints, given the resolution. The structure of the BG505 SOSIP in complex with a PGT121 family precursor (PDB accession no. 5CEZ) was used to manually build the fusion peptide of 253-11 in Coot. HXB2 numbering was used throughout. Data processing and refinement statistics are reported in Table 3. Protein structure superimpositions and center-of-mass analyses were conducted by using PyMOL v1.8.6.0 (100). The centers of mass of individual gp120 protomers (residues 32 to 504) and distances between the three gp120 centers of mass were calculated by using the “centerofmass” and “measurement” commands in PyMOL, respectively. Software were accessed through SBGrid (101).

T.M. is supported by the National Research Foundation, South Africa; the Poliomyelitis Research Foundation; and the University of Cape Town. J.E.-O. is supported by Banting postdoctoral fellowship BPF-144483 from the Canadian Institutes of Health Research (CIHR). This study was supported by the University of Cape Town (J.R.D.), the International Centre for Genetic Engineering and Biotechnology (J.R.D.), the Poliomyelitis Research Foundation (J.R.D.), and operating grant NIH-150414 (J.-P.J.) from the CIHR in partnership with the Bill and Melinda Gates Foundation. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (J.-P.J.). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. SAXS experiments were performed at the BioSAXS 18-ID-D beamline, which is supported by grant P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. X-ray diffraction data for the 253-11–10-1074 crystal described in this paper were recorded at the 23-ID-D beamline. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

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